Among the different techniques used to modify surfaces, the deposition of polyelectrolyte multilayers (PEM) has emerged as a very easy handling and versatile tool. Based on the alternate adsorption of polycations and polyanions, this technique allows to buildup films with tunable properties: by adjusting several parameters during buildup such as the chemical nature of the polycations and/or polyanions, pH and ionic strength, immersion and rinsing times, post-treatment of the film, it is possible to obtain an almost infinite variety of architectures. The introduction of electrostatic layer-by-layer (LbL) self-assembly also called electrostatic self-assembly (ESA) has shown broad biotechnology and biomedical applications in thin film coating, micropatterning, nanobioreactors, artificial cells, integrated optics, microelectronic devices, sensors, optical memory devices, encapsulation and drug delivery systems. Indeed, this kind of film is very easy to manufacture.
Of special importance for biomedical applications is the control of the chemical composition of the surface which can affect biological activity. Films made from polypeptides, i.e. poly(L-lysine) and natural polyelectrolytes (e.g., hyaluronan, alginate, chitosan, collagen) allow, for example, biomimetic architectures to be created. Applications include also the fabrication of non adhesive barriers for vascular grafts, the fabrication of films with pro- or anti-coagulant properties or the preparation of hollow capsules for drug release. Bioactivity of the films can be achieved by their functionalization by inserting peptides associated to polyelectrolytes or through the embedding of proteins. For biomaterial applications, biocompatibility is a major requirement: the material or the film covering a material surface must be non-cytotoxic to any living cell and not iatrogenic or allergenic. Another requirement is that the material possesses chemical and physical properties that promote specific cell interactions, either cell adhesion or non-adhesion depending on the final application. In this respect, it was shown that primary cells can be grown on poly(styrenesulfonate)/poly(allyamine hydrochloride) films and on poly(L-lysine)/poly(L-glutamic acid) films for several days while maintaining their phenotype. For instance, Mendelsohn et al., Biomacromolecules, 2003, 4, 96-106, showed that poly(acrylic acid)/poly(allylamine hydrochloride) multilayers can be either non adhesive or adhesive depending on the pH of preparation of the films.
Polyelectrolyte multilayers based on biopolymers or polyamino acids are hydrogels and must be considered as “soft” and sensitive materials. For example, exposure to solvents, pH and ionic strength jumps can affect their structural integrity and cross-linking constitutes a possible way to stabilize them. Up to now, only few cross-linkable PEM systems have been reported. The approaches generally rely on the cross-linking through condensation reaction of complementary groups located on adjacent layer. Recently, the inventors have described a convenient method to produce crosslinked polyelectrolyte multilayer films which prove to be stabilized with respect to aggressive media, such as solvents, extreme pH, ionic strengths jumps, enzymes and/or phagocytic cells, and can therefore withstand numerous physical, chemical and biological stresses (see EP 1,535,952 and Richert et al. Biomacromolecules, 5, 284-294, 2004; Etienne et al. Biomacromolecules. 6, 726-733, 2005; Picart et al., Adv. Funct. Mat. 15: 1771-1780, 2005).
The controlled delivery of certain proteins from a surface, in particular a biomaterial surface, is an important challenge. This would offer the possibility to concentrate the protein and deliver it locally, thereby also protecting it from degradation by enzymes in tissue fluids. The ability to design surface that can direct cell fate is also an important challenge in the field of tissue engineering and biomaterials. The surface properties of biomaterials often dictate the direct interactions of the material with its environment, in particular cellular interaction. The layer-by-layer (LbL) technique appears to be a convenient strategy for such an application as it allows the precise control of various parameters, such as film architecture, thickness, chemistry, and mechanical properties (Jessel, N. et al. Adv. Mater. 2003, 26, 692-695). As the deposition is achieved in aqueous solution, incorporation of sensitive biomolecules such as proteins is possible (Ai, H. et al. Cell Biochem. Biophys. 2003, 39, 23-43). It has been shown that proteins adsorbed on top or added as a regular layer in the LbL film retain their activity (Vodouhe, C. et al. Biomaterials 2005, 26, 6836-6845).
It is therefore an object of this invention to provide a process for coating a surface with a polyelectrolyte multilayer film incorporating a large amount of proteins, the incorporation rate of proteins being high in comparison with systems available on the market, such as collagen sponges or membranes.
It is a further object of the invention to provide a coated article by a cross-linked polyelectrolyte multilayers film directly obtained by the above mentioned process, said film comprising proteins whose bioactivity remains.
It is a further object of the invention to provide a coated article by a cross-linked polyelectrolyte multilayers film presenting a high concentration of a protein which can be delivered locally, and also protecting it from degradation by enzymes in tissue fluids.
Furthermore, it is an object of the invention to provide a coated article capable of delivering proteins with no burst effect and in particular as a slow release system, and more specifically wherein cells can adhere and differentiate in the case where proteins are growth factors.
The inventors have now discovered that cross-linked polyelectrolyte multilayers film obtained by a coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and optionally in the presence of N-hydroxysuccinimide compounds, and subsequently treated to incorporate therein a protein can be of valuable interest and presents several advantages.